Gene expression may be regulated in several ways, which include the activation or suppression of transcription, the differential processing and stabilization of messenger RNA (“mRNA”) and the extent of translation of the mRNA. The control of transcription plays a particularly critical role in the regulation of gene expression in eukaryotic cells. There are several structural elements that are involved in the regulation of transcription.
Promoters represent a class of nucleic acid structures that are involved in the regulation of transcription. In general, promoters are located next to the transcription start site and interact with RNA polymerase, either directly or indirectly. Promoters often comprise several discrete “cis elements,” each of which may be recognized by one or more trans-acting regulatory proteins known as transcription factors. Among the various cis elements well-known in the art is the “TATA box,” which is known to interact with certain regulatory proteins, e.g., transcription factors, and is generally located about 20-30 base pairs upstream from the transcription initiation site.
The binding of such transcription factors to promoters or other regulatory sequences is often responsible for the initiation, maintenance and/or down-regulation of transcription. A typical gene-specific eukaryotic transcription factor includes a DNA-binding domain and one or more additional domains that influence the activation or repression of transcription, e.g., “trans-acting domains.” Transcription factors bind in the general proximity (although occasionally at great distances) of the point of transcription initiation of a gene. Such transcription factors often act to influence the efficiency of formation or function of a transcription initiation complex at the promoter. Transcription factors can act in a positive fashion (transactivation) or in a negative fashion (transrepression). Furthermore, the effect that transcription factors may have on gene expression can be constitutive (always “on”) or conditional.
Over the years, several classes of DNA-binding domains of various transcription factors have been characterized and the nucleic acid sequences to which such domains interact identified. Non-limiting examples of such domains include motifs known as the leucine zipper, the bZIP domain, the zinc-finger, the homeobox, the basic helix-loop-helix and others. The trans-acting domains of transcription factors are often characterized as having a high content of specific amino acids, which include domains rich in acidic amino acids, proline or glutamine (Giniger et al., 1985; Meshi and Iwabuchi, 1995; Mitchell and Tjian, 1989). Acidic domains have been reported to possess activation functions that include interactions with TATA-binding proteins (“TBP”) (Truant et al., 1993), TBP-associated factors (“TAFs”) (Uesugi et al., 1997), TFIIA (Pugh, 2000), TFIIB (Klemm et al., 1995) and other general transcription complexes (Stargell and Struhl, 1995).
Beachy, in U.S. Pat. No. 5,824,857 entitled “Plant Promoter,” described the promoter from the rice tungro bacilliform virus (“RTBV”). The '857 patent discloses that the RTBV promoter causes preferential gene expression in plant vascular tissue. The patent also discloses that the RTBV promoter can be used to drive expression in most plants, whether monocotyledonous or dicotyledonous, and is particularly suited to rice. The patent further discloses the transformation of plants by inserting the coding sequence of the RTBV promoter and a heterologous gene of interest to obtain transgenic plants that express the gene of interest in vascular tissue.
Yin and Beachy, in “The regulatory regions of the rice tungro bacilliform virus promoter and interacting nuclear factors in rice (Oryza sativa L.), The Plant Journal, 7(6): 969-980 (1995),” described the E fragment (−164 to +45 in relation to the transcription start site) within the RTBV promoter, which was shown to be sufficient to cause tissue-specific gene expression. The article also disclosed a critical cis element, Box II (−53 to −39), within the E fragment that was shown to be essential for promoter activity. The same authors identified other cis elements of the RTBV promoter in “Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice, The Plant Journal: 12(5): 1179-1188 (1997),” including the ASL Box (−98 to −79) and a GATA motif (−143 to −135). Together, these cis elements were shown to confer phloem-specific reporter gene expression.
Yin et al., in “RF2a, a bZIP transcriptional activator of phloem-specific rice tungro bacilliform virus promoter, function in vascular development, The EMBO Journal, 16(17): 5247-5259 (1997),” identified a 1.8 Kb transcription factor consisting of 368 amino acids—designated as RF2a. The RF2a transcription factor is currently known to represent a bZIP transcription activator found in rice plants that contains acidic, proline-rich and glutamine-rich putative functional domains. RF2a has been shown to bind the Box II element of the RTBV promoter and stimulate Box II-dependent transcription in vitro. Another bZIP protein, RF2b, has been isolated through interaction with RF2a, which also has been shown to interact with the Box II element.
The inventors have discovered that the Box II cis-element of the RTBV promoter is portable and that it can be used to modulate gene expression in unrelated promoters in connection with RF2a and/or RF2b. That is, until now, it was not known that the Box II element, and similar sequences, could be used in chimeric promoters to regulate gene expression in connection with RF2a and/or RF2b. What's more, the inventors have discovered that the acidic domain of RF2a is particularly critical to the activation function of this transcription factor and, moreover, that it can be transferred to unrelated DNA-binding proteins to modulate gene expression. Accordingly, the inventors have discovered a new system for modulating gene expression, as described further below.